1. Field of the Invention
This invention to immunoassay of an analyte and materials used therein, and more particularly relates to a method and materials for enzyme immunoassay using delta 5-3-ketosteroid isomerase.
2. Background of the Invention
A variety of assay systems which are both rapid and sensitive has been developed to detect or determine the concentration of a substance, generally referred to as the analyte, in a liquid. Immunoassays depend on the binding of the analyte to a specific antianalyte, and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support, and are of two basic types. In competitive assays, the tracer is labeled analyte, and the analyte and tracer compete for a limited number of antianalyte binding sites. In sandwich assays, the tracer is a labeled second antianalyte specific for a second determinant on the analyte giving an antianalyte-analyte-labeled antianalyte sandwich.
Various means for labeling have been developed. Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. However, all RIA procedures require a separation step, since the parameter measured (nuclear decay) cannot be controlled by changing assay conditions or components. In addition, isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must be followed.
Fluoroimmunoassay (FIA) uses fluorochromes as labels, provides direct detection of the label, and is readily adaptable to homogeneous assay procedures. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA, largely because o light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium.
Enzymes have also been used as labels in immunoassay. Enzyme immunoassay (EIA) combines the advantages of RIA and FIA and overcomes many of the disadvantages of the other two methods. Enzyme labeled reagents are cheap to prepare and are highly stable thus giving a long shelf life, yet yield assays which approach the sensitivity of radioimmunoassay and which give objective results that can be determined, either visually, or with rather simple equipment, such as a spectrophotometer.
In conventional EIA, an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a color wich is measured. Often, an unconjugated component is immobilized on a solid support. Representative of such conventional EIA is U.S. Pat. No. 3,654,090 to Schuurs et al.
Many enzymes, such as horseradish peroxidase and alkaline phosphatase have been used as labels in EIA. U.S. Pat. No. 4,298,686 discloses use of delta 5-3-ketosteroid isomerase conjugated to an antigen as a label in a method for EIA in which ultraviolet spectroscopy is used to detect the label.
Redox reactions between delta 4-3-ketosteroids and tetrazolium salts are conventionally used in analysis of corticosteroids (the "blue tetrazolium reaction"). Discussions of this procedure are given by Meyer et al. in Anal. Chem. 27, 813 (1955) and by Graham et al. in J. Pharm. Sci. 64, 226 (1975).
An EIA method including colorimetric detection of delta 5-3-ketosteroid isomerase which would provide rapid visual detection of highly dilute analytes and avoid the need for expensive instrumentation would be desirable. The present invention is directed toward this objective.